Equilibrium non-consuming fluorescence sensor for real time intravascular glucose measurement

ABSTRACT

Embodiments of the present invention relates to analyte sensors. In particular, the preferred embodiments of the present invention relate to non-consuming intravascular glucose sensors based on fluorescence chemistry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit to U.S. Provisional No.60/917,307 filed May 10, 2007, the entirety of which is herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the present invention relates to analyte sensors. Inparticular, the preferred embodiments of the present invention relate tonon-consuming intravascular glucose sensors based on fluorescencechemistry.

2. Description of the Related Art

There has been an on-going effort over many years to use fluorescencetechniques to measure polyhydroxyl compound (e.g., glucose)concentration in bodily fluids. But despite the effort, no practicalsystem has been developed and commercialized for in vivo monitoring.Several attempts have been made to detect glucose by fluorescence usingdyes associated with boronic acid groups. Boronate moieties bind glucosereversibly. When boronic acid functionalized fluorescent dyes bindglucose, the properties of the dye are affected, such that a signalrelated to the concentration of glucose may be generated and detected.These changes have been used in the past to measure glucoseconcentration.

Russell (U.S. Pat. Nos. 5,137,833 and 5,512,246) used a boronic acidfunctionalized dye that bound glucose and generated a signal related tothe glucose concentration. James et al. (U.S. Pat. No. 5,503,770)employed a similar principle, but combined a fluorescent dye, an aminequenching functionality, and boronic acid in a single complex. Thefluorescence emission from the complex varied with the amount of glucosebinding. Van Antwerp et al. (U.S. Pat. Nos. 6,002,954 and 6,011,984)combined features of the previously cited references and also discloseda device purported to be implantable. A. E. Colvin, Jr. (U.S. Pat. No.6,304,766) also disclosed optical-based sensing devices for in situsensing in humans that utilize boronate-functionalized dyes.

Certain measurable parameters using blood or bodily fluid, such as pHand concentrations of O₂, CO₂, Na⁺, K⁺, and polyhydroxyl compounds, likeglucose, have been determined in vivo. The ability to do thesemeasurements in vivo is important because it is necessary to makefrequent determinations of such analytes when monitoring a patient.Typically, one sensor for each analyte has been placed in a patient'sblood vessel(s). If it is desired to measure several analytes, aplurality of sensors is often required, which can cause attendantdiscomfort to the patient and complexity of the electronic monitoringequipment.

In an effort to solve the design problems posed by the limitation inphysical dimension for in vivo monitoring, others have incorporateddifferent dyes into one device to get simultaneous readings of twoparameters. For example, Alder et al. (U.S. Pat. No. 5,922,612)disclosed a method for optical determination of pH and ionic strength ofan aqueous sample using two different dyes on one sensor. Gray et al.(U.S. Pat. No. 5,176,882) taught the use of a fiber optic deviceincorporating a hydrophilic polymer with immobilized pH sensitive dyeand potassium or calcium sensitive fluorescent dyes to measure theanalyte concentration in conjunction with pH. In U.S. Pat. No.4,785,814, Kane also disclosed the use of two dyes embedded in acomposite membrane for the simultaneous measurements of pH and oxygencontent in blood. However, incorporation of multiple dyes into a singlesensor complicates the manufacture of such sensors.

Besides the foregoing problems associated with separate indwellingsensors for each analyte being monitored, particularly in the intensivecare setting, and multiple dye sensors, another problem associated withmany dye-based analyte sensors is pH sensitivity. A slight change in pHmay modify or attenuate fluorescence emissions, and cause inaccuratereadings. This problem is particularly acute for monitoring bloodglucose levels in diabetic patients, whose blood pH may fluctuaterapidly. Since accurate blood glucose level measurements are essentialfor treating these patients, there is a significant need for a glucosesensor that facilitates real-time correction of the pH effect withoutrequiring separate indwelling pH and analyte sensors, or sensors havingmultiple dyes.

Ratiometric pH determination using fluorescent dye(s) is known. Given afluorophore that has an acid and base form, the ratio of the emissionintensity of the two forms can be used as a measure of the pH that isinsensitive to fluorophore concentration . See e.g., U.S. PatentPublication No. 2005/0090014 which describes an HPTS-derived pHsensitive dye (incorporated herein in its entirety by reference); Niu C.G. et al. 2005 Anal. Bioanal. Chem. 383(2):349-357, which describes apH-sensitive dye meso-5,10,15,20-tetra-(4-allyloxyphenyl)porphyrin(TAPP) as an indicator, and a pH-insensitive benzothioxanthenederivative as a reference, for fluorescence ratiometric measurement;Turner N. G. et al. 1998 J. Investig. Dermatol. Symp. Proc. August3(2):110-3, which discloses dual-emission ratiometric imaging using thefluorophore, carboxy seminaphthorhodafluor-1, which displays apH-dependent shift in its emission spectrum; and Badugu R. et al. 2005Talanta 66:569-574, which describes the use of 6-aminoquinoliniumboronic acid dyes that show spectral shifts and intensity changes withpH in a wavelength-ratiometric manner.

However, despite the inventor's recognition of a substantial unmet needfor a sensor adapted to provide continuous intravascular monitoring ofpH and glucose, wherein the glucose measurement may be corrected for pHeffects, no one has disclosed or even suggested using a sensorcomprising a single fluorophore that exhibits properties suitable tomake a ratiometric pH measurement that is independent of the fluorophoreconcentration, where the same fluorophore is functionalized to bindglucose and generate a signal the intensity of which is related to theglucose concentration.

SUMMARY OF THE INVENTION

An intravascular sensor is disclosed in accordance with preferredembodiments of the present invention for determining the analyteconcentration in blood. The sensor comprises an analyte bindingmolecule, wherein the analyte binding molecule is associated with afluorophore; an analyte analog, wherein the analyte analog is associatedwith an acceptor, wherein the fluorophore is capable of emittingradiation at a wavelength that is absorbed at least in part by theacceptor, wherein the fluorophore is capable of transferring energy tothe acceptor via fluorescence resonance energy transfer when thefluorophore is in close proximity to the acceptor; a light source; and adetector.

In some embodiments, the sensor further comprises a hydrogel, whereinthe analyte binding molecule and the analyte analog are substantiallyimmobilized in the hydrogel, wherein the hydrogel is permeable to theanalyte.

In some embodiments, the light source is a laser.

In some embodiments, the laser is capable of delivering a frequencymodulated excitation signal.

In some embodiments, the laser is capable of delivery a pulse of light.

In some embodiments, the sensor further comprises an optical fiber.

In some embodiments, the analyte is glucose.

A method is disclosed in accordance with preferred embodiments of thepresent invention for determining the glucose concentration in blood.The method comprises providing the sensor as described above; insertingthe sensor into a blood vessel; irradiating the fluorophore with anexcitation signal; detecting a fluorescence emission from thefluorophore; and determining the concentration of glucose.

In some embodiments, the excitation signal is a pulse of light.

In some embodiments, the method further comprises measuring the decay ofthe fluorescence emission over time and calculating a fluorescencelifetime.

In some embodiments, the excitation signal is frequency modulated.

In some embodiments, the method further comprises measuring the phaseshift between the emission and the excitation signal.

In some embodiments, the method further comprises calculating afluorescence lifetime.

In some embodiments, the method further comprises measuring a modulationratio and calculating a fluorescence lifetime.

In one preferred embodiment, an intravascular sensor is disclosed fordetermining an analyte concentration in blood. The sensor comprises anoptical fiber comprising a sensor chemistry portion disposed along adistal region of the optical fiber, wherein the sensor chemistry portionis sized and physiologically compatible with residing in a blood vessel,wherein the sensor chemistry portion is selected from equilibriumfluorescence chemistry or lifetime chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing the sensing mechanism of one embodimentof the present invention.

FIG. 2 shows a glucose and pH sensor and optical system comprising twoexcitation light sources and two detectors in accordance with onepreferred embodiment of the present invention.

FIG. 3 shows the absorption spectra of HPTS at different pHs.

FIG. 4 shows independence of ratiometric pH sensing using HPTS/MABP4using the I_((base))/I_((iso)) ratio from glucose concentration. Thedata are plotted as a ratio of the fluorescence emission forcorresponding to excitation at 454 nm (base) and 422 nm (isobesticpoint) vs. pH in various glucose concentrations.

FIG. 5 shows glucose response curves for HPTS/MABP4 excited at 422 nm(isobestic point) at different pHs.

FIG. 6 shows the absorption spectra of SNARF-1 at different pHs insolution.

FIG. 7 shows glucose response curves. for SNARF-1/3,3′-oBBV in solutionat different pHs excited at 514 nm/emission at 587 nm.

FIG. 8 shows glucose response curves for SNARF-1/3,3′-oBBV in solutionat different pHs excited at 514 nm/emission at 625 nm.

FIG. 9 shows ratiometric sensing of pH at different glucoseconcentrations with SNARF-1/3,3′-oBBV in solution using theI_((base))/I_((acid)) ratio.

FIG. 10 shows glucose response curves for HPTS-triLysMA/3,3′-oBBV/DMAAat different pHs.

FIG. 11 shows ratiometric sensing of pH at different glucoseconcentrations using HPTS-triLysMA/3,3′-oBBV/DMAA, using theI_((base))/I_((acid)) ratio.

FIG. 12 shows ratiometric sensing of pH at different glucoseconcentrations using HPTS-triCysMA/3,3′-oBBV/DMMA wherein the indicatorsystem is immobilized on the end of an optical fiber, using theI_((base))/I_((acid)) ratio.

FIG. 13 shows a graph of the decay of fluorescence intensity over timeafter a fluorophore is subjected to a pulse of excitation light.

FIG. 14 shows a graph of the emission signal resulting from a frequencymodulated excitation signal.

FIGS. 15A-C show the interaction between a glucose binding moleculelinked to a fluorophore, a glucose analog linked to an acceptor and aglucose molecule.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In a preferred embodiment, the present invention is directed to anoptical sensor capable of measuring two analytes with a single indicatorsystem. More particularly, the preferred sensor employs a singlefluorophore (e.g., a fluorescent dye) to: (1) determine theconcentration of a first analyte, e.g., H⁺ (pH), by a ratiometricmethod, wherein such determination is independent of the concentrationof the fluorophore; and (2) determine the concentration of a secondanalyte, e.g., a polyhydroxyl compounds (e.g., preferably glucose) bymeasuring the apparent fluorophore concentration (e.g., emissionintensity of the fluorophore upon excitation), wherein the apparentfluorophore concentration is dependent on the concentration of thesecond analyte. Further, where measurement of the second analyteconcentration is dependent on the first analyte concentration (e.g., inoptical systems in which glucose measurement varies with pH—a commonproblem in this field), then in accordance with a preferred embodimentof the present invention, the measured second analyte concentration maybe corrected for the contribution of the first analyte concentration.The sensor is preferably stable in aqueous media (e.g., physiologicalmedia, blood, interstitial fluid, etc.), and more preferably, the sensoris configured to be inserted into a blood vessel where it can remainindwelling for a period of time. Thus, in accordance with a preferredembodiment of the present invention, an optical sensor configured forintravascular placement is disclosed, which sensor is capable ofmeasuring two analytes (preferably pH and glucose) with a singleindicator system and correcting the glucose measurement for anycontributions of pH.

Although preferred embodiments of the sensor are directed inter alia toratiometric pH sensing, other first analyte concentrations may bedetermined in accordance with the broader scope of the presentinvention, as long as the indicator system comprises a fluorophore thatexists in at least two forms the concentration of which are associatedwith the concentration of the first analyte and the emission ratio ofwhich is independent of the fluorophore concentration. Likewise,although glucose is used as a second analyte example herein, it isunderstood that the concentration of other polyhydroxyl-containingorganic compounds (carbohydrates, 1,2-diols, 1,3-diols and the like) ina solution may be determined using embodiments of this invention, aslong as the indicator system comprises a fluorophore that is operablycoupled to a binding moiety that binds the second analyte, wherein thesignal intensity of the fluorophore varies with the concentration ofsecond analyte. In some embodiments, the concentration of secondanalytes may including non-carbohydrates.

Indicator System

The indicator systems used in accordance with preferred embodiments ofthe present invention comprise a fluorophore operably coupled to ananalyte binding moiety, wherein analyte binding causes an apparentoptical change in the fluorophore concentration (e.g., emissionintensity). It is further desired that the fluorophore has differentacid and base forms that exhibit a detectable difference in spectralproperties such that ratiometric pH sensing may be enabled. For example,a glucose binding moiety such as 3,3′-oBBV (described in detail below)that is operably coupled to a fluorescent dye such as HPTS-triLysMA(described in detail below) will quench the emission intensity of thefluorescent dye, wherein the extent of quenching is reduced upon glucosebinding resulting in an increase in emission intensity related toglucose concentration. In preferred embodiments, the indicator systemscomprise a dye having at least two anionic groups and a quencher havingat least two boronic acids. In further preferred embodiments, theindicator systems also comprise a means for immobilizing the sensingmoieties (e.g., dye-quencher) such that they remain physically closeenough to one another to react (quenching). Where in vivo sensing isdesired, such immobilizing means are preferably insoluble in an aqueousenvironment (e.g., intravascular), permeable to the target analytes, andimpermeable to the sensing moieties. Typically, the immobilizing meanscomprises a water-insoluble organic polymer matrix. For example, theHPTS-triLysMA dye and 3,3′-oBBV quencher may be effectively immobilizedwithin a DMAA (N,N-dimethylacrylamide) hydrogel matrix (described indetail below), which allows pH and glucose sensing in vivo.

Some exemplary and preferred fluorophores, analyte binding moieties andimmobilizing means are set forth in greater detail below.

Fluorophores

“Fluorophore” refers to a substance that when illuminated by light at aparticular wavelength emits light at a longer wavelength; i.e. itfluoresces. Fluorophores include but are not limited to organic dyes,organometallic compounds, metal chelates, fluorescent conjugatedpolymers, quantum dots or nanoparticles and combinations of the above.Fluorophores may be discrete moieties or substituents attached to apolymer.

Fluorophores that may be used in preferred embodiments are capable ofbeing excited by light of wavelength at or greater than about 400 nm,with a Stokes shift large enough that the excitation and emissionwavelengths are separable by at least 10 nm. In some embodiments, theseparation between the excitation and emission wavelengths may be equalto or greater than about 30 nm. These fluorophores are preferablysusceptible to quenching by electron acceptor molecules, such asviologens, and are resistant to photo-bleaching. They are alsopreferably stable against photo-oxidation, hydrolysis andbiodegradation.

In some embodiments, the fluorophore may be a discrete compound.

In some embodiments, the fluorophore may be a pendant group or a chainunit in a water-soluble or water-dispersible polymer having molecularweight of about 10,000 daltons or greater, forming a dye-polymer unit.In one embodiment, such dye-polymer unit may also be non-covalentlyassociated with a water-insoluble polymer matrix M¹ and is physicallyimmobilized within the polymer matrix M¹, wherein M¹ is permeable to orin contact with analyte solution. In another embodiment, the dye on thedye-polymer unit may be negatively charged, and the dye-polymer unit maybe immobilized as a complex with a cationic water-soluble polymer,wherein said complex is permeable to or in contact with the analytesolution. In one embodiment, the dye may be one of the polymericderivatives of hydroxypyrene trisulfonic acid. The polymeric dyes may bewater-soluble, water-swellable or dispersible in water. In someembodiments, the polymeric dyes may also be cross-linked. In preferredembodiments, the dye has a negative charge.

In other embodiments, the dye molecule may be covalently bonded to thewater-insoluble polymer matrix M¹, wherein said M¹ is permeable to or incontact with the analyte solution. The dye molecule bonded to M¹ mayform a structure M¹-L¹-Dye. L¹ is a hydrolytically stable covalentlinker that covalently connects the sensing moiety to the polymer ormatrix. Examples of L¹ include lower alkylene (e.g., C₁-C₈ alkylene),optionally terminated with or interrupted by one or more divalentconnecting groups selected from sulfonamide (—SO₂NH—), amide —(C═O)N—,ester —(C═O)—O—, ether. —O—, sulfide —S—, sulfone (—SO₂—), phenylene—C₆H₄—, urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—,amide —(C═O)NH—, amine —NR— (where R is defined as alkyl having 1 to 6carbon atoms) and the like, or a combination thereof. In one embodiment,the dye is bonded to a polymer matrix through the sulfonamide functionalgroups.

In some embodiments, useful dyes include pyranine derivatives (e.g.hydroxypyrene trisulfonamide derivatives and the like), which have thefollowing formula:

wherein R¹, R², R³ are each —NHR⁴, R⁴ is —CH₂CH₂(—OCH₂CH₂—)_(n)X¹;wherein X¹ is —OH, —OCH₃COOH, —CONH₂, —SO₃H, —NH₂, or OMe; and n isbetween about 70 and 10,000. In one embodiment, the dyes may be bondedto a polymer through the sulfonamide functional groups. In otherembodiments, the dye may be one of the polymeric derivatives ofhydroxypyrene trisulfonic acid.

In some embodiments, the fluorescent dye may be8-hydroxypyrene-1,3,6-trisulfonate (HPTS). The counterions can be H⁺ orany other cation. HPTS exhibits two excitation wavelengths at around 450nm and around 405 nm, which correspond to the absorption wavelengths ofthe acid and its conjugate base. The shift in excitation wavelength isdue to the pH-dependent ionization of the hydroxyl group on HPTS. As thepH increases, HPTS shows an increase in absorbance at about 450 nm, anda decrease in absorbance below about 420 nm. The pH-dependent shift inthe absorption maximum enables dual-excitation ratiometric detection inthe physiological range. This dye has a molecular weight of less than500 daltons, so it will not stay within the polymer matrix, but it canbe used with an anion exclusion membrane.

In another embodiment, the fluorescent dye may be polymers of8-acetoxy-pyrene-1,3,6-N,N′,N″-tris-(methacrylpropylamidosulfonamide)(acetoxy-HPTS-MA):

It is noted that dyes such as acetoxy-HPTS-MA (above) having no anionicgroups, may not give very strong glucose response when operably coupledto a viologen quencher, particularly a viologen quencher having only asingle boronic acid moiety.

In another embodiment, the fluorescent dye may be8-hydroxy-pyrene-1,3,6-N,N′,N″-tris-(carboxypropylsulfonamide)(HPTS-CO₂):

In another embodiment, the fluorescent dye may be8-hydroxy-pyrene-1,3,6-N,N′,N″-tris-(methoxypolyethoxyethyl (˜125)sulfonamide) (HPTS-PEG):

It is noted that dyes such as HPTS-PEG (above) having no anionic groups,may not provide a very strong glucose response when operably coupled toa viologen quencher, particularly a viologen quencher having only asingle boronic acid moiety.

Representative dyes as discrete compounds are the tris adducts formed byreacting 8-acetoxypyrene-1,3,6-trisulfonylchloride (HPTS-Cl) with anamino acid, such as amino butyric acid. Hydroxypyrene trisulfonamidedyes bonded to a polymer and bearing one or more anionic groups are mostpreferred, such as copolymers of8-hydroxypyrene-1-N-(methacrylamidopropylsulfonamido)-N′,N″-3,6-bis(carboxypropylsulfonamide)HPTS-CO₂-MA with HEMA, PEGMA, and the like.

In another embodiment, the fluorescent dye may be HPTS-TriCys-MA:

This dye may be used with a quencher comprising boronic acid, such as3,3′-oBBV.

Of course, in some embodiments, substitutions other than Cys-MA on theHPTS core are consistent with aspects of the present invention, as longas the substitutions are negatively charged and have a polymerizablegroup. Either L or D stereoisomers of cysteine may be used. In someembodiments, only one or two of the sulfonic acids may be substituted.Likewise, in variations to HPTS-CysMA shown above, other counterionsbesides NBu₄ ⁺ may be used, including positively charged metals, e.g.,Na⁺. In other variations, the sulfonic acid groups may be replaced withe.g., phosphoric, carboxylic, etc. functional groups.

Another suitable dye is HPTS-LysMA, which is pictured below as follows:

Other examples include soluble copolymers of 8-acetoxypyrene-1,3,6-N,N′,N″-tris(methacrylamidopropylsulfonamide) with HEMA, PEGMA, or otherhydrophilic comonomers. The phenolic substituent in the dye is protectedduring polymerization by a blocking group that can be removed byhydrolysis after completion of polymerization. Such suitable blockinggroups, as for example, acetoxy, trifluoroacetoxy, and the like, arewell known in the art.

Fluorescent dyes, including HPTS and its derivatives are known and manyhave been used in analyte detection. See e.g., U.S. Pat. Nos. 6,653,141,6,627,177, 5,512,246, 5,137,833, 6,800,451, 6,794,195, 6,804,544,6,002,954, 6,319,540, 6,766,183, 5,503,770, and 5,763,238; andco-pending U.S. patent application Ser. Nos. 11/296,898 and 60/833,081;each of which is incorporated herein in its entirety by referencethereto.

The SNARF and SNAFL dyes from Molecular Probes may also be usefulfluorophores in accordance with aspects of the present invention. Thestructures of SNARF-1 and SNAFL-1 are shown below.

Additionally, a set of isomeric water-soluble fluorescent probes basedon both the 6-aminoquinolinium and boronic acid moieties which showspectral shifts and intensity changes with pH, in awavelength-ratiometric and calorimetric manner may be useful inaccordance with some embodiments of the present invention (See e.g.,Badugu, R. et al. 2005 Talanta 65 (3):762-768; and Badugu, R. et al.2005 Bioorg. Med. Chem. 13 (1):113-119); incorporated herein in itsentirety by reference.

Another example of a fluorescence dye that may be pH and saccharidesensitive is tetrakis(4-sulfophenyl)porphine (TSPP)-shown below. TSPPmay not work optimally in blood, where the porphyrin ring may react withcertain metal ions, like ferric, and become non-fluorescent.

Additional examples of pH sensitive fluorescent indicators that may beuseful for simultaneous determination of pH and glucose in the sensor ofthe present invention are described in US 2005/0233465 and US2005/0090014; each of which is incorporated herein by reference in itsentirety.

Analyte Binding Moieties—Quenchers

In accordance with broad aspects of the present invention, the analytebinding moiety provides the at least dual functionality of being able tobind analyte and being able to modulate the apparent concentration ofthe fluorophore (e.g., detected as a change in emission signalintensity) in a manner related to the amount of analyte binding. Inpreferred embodiments, the analyte binding moiety is associated with aquencher. “Quencher” refers to a compound that reduces the emission of afluorophore when in its presence. Quencher (Q) is selected from adiscrete compound, a reactive intermediate which is convertible to asecond discrete compound or to a polymerizable compound or Q is apendant group or chain unit in a polymer prepared from said reactiveintermediate or polymerizable compound, which polymer is water-solubleor dispersible or is an insoluble polymer, said polymer is optionallycrosslinked.

In one example, the moiety that provides glucose recognition in theembodiments is an aromatic boronic acid. The boronic acid is covalentlybonded to a conjugated nitrogen-containing heterocyclic aromaticbis-onium structure (e.g., a viologen). “Viologen” refers generally tocompounds having the basic structure of a nitrogen containing conjugatedN-substituted heterocyclic aromatic bis-onium salt, such as 2,2′-, 3,3′-or 4,4′-N,N′bis-(benzyl) bipyridium dihalide (i.e., dichloride, bromidechloride), etc. Viologen also includes the substituted phenanthrolinecompounds. The boronic acid substituted quencher preferably has a pKa ofbetween about 4 and 9, and reacts reversibly with glucose in aqueousmedia at a pH from about 6.8 to 7.8 to form boronate esters. The extentof reaction is related to glucose concentration in the medium. Formationof a boronate ester diminishes quenching of the fluorphore by theviologen resulting in an increase in fluorescence dependent on glucoseconcentration. A useful bis-onium salt is compatible with the analytesolution and capable of producing a detectable change in the fluorescentemission of the dye in the presence of the analyte to be detected.

Bis-onium salts in the embodiments of this invention are prepared fromconjugated heterocyclic aromatic di-nitrogen compounds. The conjugatedheterocyclic aromatic di-nitrogen compounds are selected fromdipyridyls, dipyridyl ethylenes, dipyridyl phenylenes, phenanthrolines,and diazafluorenes, wherein the nitrogen atoms are in a differentaromatic ring and are able to form an onium salt. It is understood thatall isomers of said conjugated heterocyclic aromatic di-nitrogencompounds in which both nitrogens can be substituted are useful in thisinvention. In one embodiment, the quencher may be one of the bis-oniumsalts derived from 3,3′-dipyridyl, 4,4′-dipyridyl and4,7-phenanthroline.

In some embodiments, the viologen-boronic acid adduct may be a discretecompound having a molecular weight of about 400 daltons or greater. Inother embodiments, it may also be a pendant group or a chain unit of awater-soluble or water-dispersible polymer with a molecular weightgreater than about 10,000 daltons. In one embodiment, thequencher-polymer unit may be non-covalently associated with a polymermatrix and is physically immobilized therein. In yet another embodiment,the quencher-polymer unit may be immobilized as a complex with anegatively charge water-soluble polymer.

In other embodiments, the viologen-boronic acid moiety may be a pendantgroup or a chain unit in a crosslinked, hydrophilic polymer or hydrogelsufficiently permeable to the analyte (e.g., glucose) to allowequilibrium to be established.

In other embodiments, the quencher may be covalently bonded to a secondwater-insoluble polymer matrix M², which can be represented by thestructure M²-L²-Q. L² is a linker selected from the group consisting ofa lower alkylene (e.g., C₁-C₈ alkylene), sulfonamide, amide, quaternaryammonium, pyridinium, ester, ether, sulfide, sulfone, phenylene, urea,thiourea, urethane, amine, and a combination thereof. The quencher maybe linked to M² at one or two sites in some embodiments.

For the polymeric quencher precursors, multiple options are availablefor attaching the boronic acid moiety and a reactive group which may bea polymerizable group or a coupling group to two different nitrogens inthe heteroaromatic centrally located group. These are:

-   -   a) a reactive group on a first aromatic moiety is attached to        one nitrogen and a second aromatic group containing at least one        —B(OH)2 group is attached to the second nitrogen;    -   b) one or more boronic acid groups are attached to a first        aromatic moiety which is attached to one nitrogen and one        boronic acid and a reactive group are attached to a second        aromatic group which second aromatic group is attached to the        second nitrogen;    -   c) one boronic acid group and a reactive group are attached to a        first aromatic moiety which first aromatic group is attached to        one nitrogen, and a boronic acid group and a reactive group are        attached to a second aromatic moiety which is attached to the        second nitrogen; and    -   d) one boronic acid is attached to each nitrogen and a reactive        group is attached to the heteroaromatic ring.

Preferred embodiments comprise two boronic acid moieties and onepolymerizable group or coupling group wherein the aromatic group is abenzyl substituent bonded to the nitrogen and the boronic acid groupsare attached to the benzyl ring and may be in the ortho- meta orpara-positions.

In some embodiments, the boronic acid substituted viologen as a discretecompound useful for in vitro sensing may be represented by one of thefollowing formulas:

-   -   where n=1-3, X is halogen, and Y¹ and Y² are independently        selected from phenyl boronic acid (o- m- or p-isomers) and        naphthyl boronic acid. In other embodiments, the quencher may        comprise a boronic acid group as a substituent on the        heterocyclic ring of a viologen.

A specific example used with TSPP is m-BBV:

The quencher precursors suitable for making sensors may be selected fromthe following:

The quencher precursor 3,3′-oBBV may be used with HPTS-LysMA orHPTS-CysMA to make hydrogels in accordance with preferred aspects of theinvention.

Preferred quenchers are prepared from precursors comprising viologensderived from 3,3′-dipyridyl substituted on the nitrogens withbenzylboronic acid groups and at other positions on the dipyridyl ringswith a polymerizable group or a coupling group. Representative viologensinclude:

-   -   where L is L1 or L2 and is a linking group    -   Z is a reactive group; and    -   R′ is —B(OH)₂ in the ortho- meta- or para-positions on the        benzyl ring and R″ is H—; or optionally R″ is a coupling group        as is defined herein or a substituent specifically used to        modify the acidity of the boronic acid such as fluoro- or        methoxy;    -   L is a divalent moiety that covalently connects the sensing        moiety to a reactive group that is used to bind the viologen to        a polymer or matrix. Examples of L include those which are each        independently selected from a direct bond or, a lower alkylene        having 1 to 8 carbon atoms, optionally terminated with or        interrupted by one or more divalent connecting groups selected        from sulfonamide (—SO₂NH—), amide —(C═O)N—, ester —(C═O)—O—,        ether —O—, sulfide —S—, sulfone (—SO₂—), phenylene —C₆H₄-,        urethane —NH(C═O)—O—, urea —NH(C═O)NH—, thiourea —NH(C═S)—NH—,        amide —(C═O)NH—, amine —NR— (where R is defined as alkyl having        1 to 6 carbon atoms) and the like.    -   Z is either a polymerizable ethylenically unsaturated group        selected from but not limited to methacrylamido-, acrylamido-,        methacryloyl-, acryloyl-, or styryl- or optionally Z is a        reactive functional group, capable of forming a covalent bond        with a polymer or matrix. Such groups include but are not        limited to —Br, —OH, —SH, —CO₂H, and —NH₂.

Boronic acid substituted polyviologens are another class of preferredquenchers. The term polyviologen includes: a discrete compound comprisedof two or more viologens covalently bonded together by a linking group,a polymer comprised of viologen repeat units in the chain, a polymerwith viologen groups pendant to the chain, a dendrimer comprised ofviologen units, preferably including viologen terminal groups, anoligomer comprised of viologen units, preferably including viologenendgroups, and combinations thereof. Polymers in which mono-viologengroups form a minor component are not included. The preferred quenchersare water soluble or dispersible polymers, or crosslinked, hydrophilicpolymers or hydrogels sufficiently permeable to glucose to function aspart of a sensor. Alternatively the polyviologen boronic acid may bedirectly bonded to an inert substrate.

A polyviologen quencher as a polymer comprised of viologen repeat unitshas the formula:

In another embodiment, the polyviologen boronic acid adducts are formedby covalently linking two or more viologen/boronic acid intermediates.The bridging group is typically a small divalent radical bonded to onenitrogen in each viologen, or to a carbon in the aromatic ring of eachviologen, or one bond may be to a ring carbon in one viologen and to anitrogen in the other. Two or more boronic acid groups are attached tothe polyviologen. Optionally, the polyviologen boronic acid adduct issubstituted with a polymerizable group or coupling group attacheddirectly to the viologen or to the bridging group. Preferably thepolyviologen moiety includes only one such group. Preferably, thebridging group is selected to enhance cooperative binding of the boronicacids to glucose.

The coupling moiety is a linking group as defined previously with theproviso that the linking group is optionally further substituted with aboronic acid, a polymerizable group, an additional coupling group, or isa segment in a polymer chain in which the viologen is a chain unit, apendant group, or any combination thereof.

Immobilizing Means

In some embodiments, for use in vitro not involving a moving stream, thesensing components are used as individual (discrete) components. The dyeand quencher are mixed together in liquid solution, analyte is added,the change in fluorescence intensity is measured, and the components arediscarded. Polymeric matrices that can be used to trap the sensingcomponents to prevent leaching need not be present. Optionally, thesensing components are immobilized which allows their use to measureanalytes in a moving stream.

For in vivo applications, the sensor is used in a moving stream ofphysiological fluid which contains one or more polyhydroxyl organiccompounds or is implanted in tissue such as muscle which contains saidcompounds. Therefore, it is preferred that none of the sensing moietiesescape from the sensor assembly. Thus, for use in vivo, the sensingcomponents are preferably part of an organic polymer sensing assembly.Soluble dyes and quenchers can be confined by a semi-permeable membranethat allows passage of the analyte but blocks passage of the sensingmoieties. This can be realized by using as sensing moieties solublemolecules that are substantially larger than the analyte molecules(molecular weight of at least twice that of the analyte or greater than1000 preferably greater than 5000); and employing a selectivesemipermeable membrane such as a dialysis or an ultrafiltration membranewith a specific molecular weight cutoff between the two so that thesensing moieties are quantitatively retained.

Preferably the sensing moieties are immobilized in an insoluble polymermatrix, which is freely permeable to glucose. The polymer matrix iscomprised of organic, inorganic or combinations of polymers thereof. Thematrix may be composed of biocompatible materials. Alternatively, thematrix is coated with a second biocompatible polymer that is permeableto the analytes of interest.

The function of the polymer matrix is to hold together and immobilizethe fluorophore and quencher moieties while at the same time allowingcontact with the analyte, and binding of the analyte to the boronicacid. To achieve this effect, the matrix must be insoluble in themedium, and in close association with it by establishing a high surfacearea interface between matrix and analyte solution. For example, anultra-thin film or microporous support matrix is used. Alternatively,the matrix is swellable in the analyte solution, e.g. a hydrogel matrixis used for aqueous systems. In some instances, the sensing polymers arebonded to a surface such as the surface of a light conduit, orimpregnated in a microporous membrane. In all cases, the matrix must notinterfere with transport of the analyte to the binding sites so thatequilibrium can be established between the two phases. Techniques forpreparing ultra-thin films, microporous polymers, microporous sol-gels,and hydrogels are established in the art. All useful matrices aredefined as being analyte permeable.

Hydrogel polymers are used in some embodiments. The term, hydrogel, asused herein refers to a polymer that swells substantially, but does notdissolve in water. Such hydrogels may be linear, branched, or networkpolymers, or polyelectrolyte complexes, with the proviso that theycontain no soluble or leachable fractions. Typically, hydrogel networksare prepared by a crosslinking step, which is performed on water-solublepolymers so that they swell but do not dissolve in aqueous media.Alternatively, the hydrogel polymers are prepared by copolymerizing amixture of hydrophilic and crosslinking monomers to obtain a waterswellable network polymer. Such polymers are formed either by additionor condensation polymerization, or by combination process. In thesecases, the sensing moieties are incorporated into the polymer bycopolymerization using monomeric derivatives in combination withnetwork-forming monomers. Alternatively, reactive moieties are coupledto an already prepared matrix using a post polymerization reaction. Saidsensing moieties are units in the polymer chain or pendant groupsattached to the chain.

The hydrogels useful in this invention are also monolithic polymers,such as a single network to which both dye and quencher are covalentlybonded, or multi-component hydrogels. Multi-component hydrogels includeinterpenetrating networks, polyelectrolyte complexes, and various otherblends of two or more polymers to obtain a water swellable composite,which includes dispersions of a second polymer in a hydrogel matrix andalternating microlayer assemblies.

Monolithic hydrogels are typically formed by free radicalcopolymerization of a mixture of hydrophilic monomers, including but notlimited to HEMA, PEGMA, methacrylic acid, hydroxyethyl acrylate, N-vinylpyrrolidone, acrylamide, N,N′-dimethyl acrylamide, and the like; ionicmonomers include methacryloylaminopropyl trimethylammonium chloride,diallyl dimethyl ammonium. chloride, vinyl benzyl trimethyl ammoniumchloride, sodium sulfopropyl methacrylate, and the like; crosslinkersinclude ethylene dimethacrylate, PEGDMA, trimethylolpropane triacrylate,and the like. The ratios of monomers are chosen to optimize networkproperties including permeability, swelling index, and gel strengthusing principles well established in the art. In one embodiment, the dyemoiety is derived from an ethylenically unsaturated derivative of a dyemolecule, such as8-acetoxypyrene-1,3,6-N,N′,N″-tris(methacrylamidopropylsulfonamide), thequencher moiety is derived from an ethylenically unsaturated viologensuch as 4-N-(benzyl-3-boronic acid)-4′-N′-(benzyl-4ethenyl)-dipyridiniumdihalide (m-SBBV) and the matrix is made from HEMA and PEGDMA. Theconcentration of dye is chosen to optimize emission intensity. The ratioof quencher to dye is adjusted to provide sufficient quenching toproduce the desired measurable signal.

In some embodiments, a monolithic hydrogel is formed by a condensationpolymerization. For example, acetoxy pyrene trisulfonyl chloride isreacted with an excess of PEG diamine to obtain a tris-(amino PEG)adduct dissolved in the unreacted diamine. A solution of excesstrimesoyl chloride and an acid acceptor is reacted with4-N-(benzyl-3-boronic acid)-4′-N′-(2 hydroxyethyl) bipyridinium dihalideto obtain an acid chloride functional ester of the viologen. The tworeactive mixtures are brought into contact with each other and allowedto react to form the hydrogel, e.g. by casting a thin film of onemixture and dipping it into the other.

In other embodiments, multi-component hydrogels wherein the dye isincorporated in one component and the quencher in another are preferredfor making the sensor of this invention. Further, these systems areoptionally molecularly imprinted to enhance interaction betweencomponents and to provide selectivity for glucose over other polyhydroxyanalytes. Preferably, the multicomponent system is an interpenetratingpolymer network (IPN) or a semi-interpenetrating polymer network(semi-IPN).

The IPN polymers are typically made by sequential polymerization. First,a network comprising the quencher is formed. The network is then swollenwith a mixture of monomers including the dye monomer and a secondpolymerization is carried out to obtain the IPN hydrogel.

The semi-IPN hydrogel is formed by dissolving a soluble polymercontaining dye moieties in a mixture of monomers including a quenchermonomer and polymerizing the mixture. In some embodiments, the sensingmoieties are immobilized by an insoluble polymer matrix which is freelypermeable to polyhydroxyl compounds. Additional details on hydrogelsystems have been disclosed in US Patent Publications Nos.US2004/0028612, and 2006/0083688 which are hereby incorporated byreference in their entireties.

The polymer matrix is comprised of organic, inorganic or combinations ofpolymers thereof. The matrix may be composed of biocompatible materials.Alternatively, the matrix is coated with a second biocompatible polymerthat is permeable to the analytes of interest. The function of thepolymer matrix is to hold together and immobilize the fluorescent dyeand quencher moieties while at the same time allowing contact with theanalytes (e.g., polyhydroxyl compounds, H⁺ and OH⁻), and binding of thepolyhydroxyl compounds to the boronic acid. Therefore, the matrix isinsoluble in the medium and in close association with it by establishinga high surface area interface between matrix and analyte solution. Thematrix also does not interfere with transport of the analyte to thebinding sites so that equilibrium can be established between the twophases. In one embodiment, an ultra-thin film or microporous supportmatrix may be used. In another embodiment, the matrix that is swellablein the analyte solution (e.g. a hydrogel matrix) can be used for aqueoussystems. In some embodiments, the sensing polymers are bonded to asurface such as the surface of a light conduit, or impregnated in amicroporous membrane. Techniques for preparing ultra-thin films,microporous polymers, microporous sol-gels, and hydrogels have beenestablished in the prior art.

In one preferred embodiment, the boronic acid substituted viologen maybe covalently bonded to a fluorescent dye. The adduct may be apolymerizable compound or a unit in a polymer. One such adduct forexample may be prepared by first forming an unsymmetrical viologen from4,4′-dipyridyl by attaching a benzyl-3-boronic acid group to onenitrogen and an aminoethyl group to the other nitrogen atom. Theviologen is condensed sequentially first with8-acetoxy-pyrene-1,3,6-trisulfonyl chloride in a 1:1 mole ratio followedby reaction with excess PEG diamine to obtain a prepolymer mixture. Anacid acceptor is included in both steps to scavange the byproduct acid.The prepolymer mixture is crosslinked by reaction with a polyisocyanateto obtain a hydrogel. The product is treated with base to remove theacetoxy blocking group. Incomplete reaction products and unreactedstarting materials are leached out of the hydrogel by exhaustiveextraction with deionized water before further use. The product isresponsive to glucose when used as the sensing component as describedherein.

Alternatively, such adducts are ethylenically unsaturated monomerderivatives. For example, dimethyl bis-bromomethyl benzene boronate isreacted with excess 4,4′-dipyridyl to form a half viologen adduct. Afterremoving the excess dipyridyl, the adduct is further reacted with anexcess of bromoethylamine hydrochloride to form the bis-viologen adduct.This adduct is coupled to a pyranine dye by reaction with the8-acetoxypyrene-tris sulfonyl chloride in a 1:1 mole ratio in thepresence of an acid acceptor followed by reaction with excessaminopropylmethacrylamide. Finally, any residual amino groups may bereacted with methacrylol chloride. After purification, the dye/viologenmonomer may be copolymerized with HEMA and PEGDMA to obtain a hydrogel.

Ratiometric pH Sensing

Ratiometric pH sensing is known. See e.g., US Pat. Publication Nos.2006/0105174; 2005/0090014; incorporated herein in their entirety byreference. Given an indicator system comprising a fluorophore (e.g., afluorescent indicator dye) that exists in two forms (an acid form and abase form) the ratio of the emission intensity at the two wavelengthscan be used to measure pH independent of the fluorophore concentration.The fluorescent indicator dyes suitable for ratiometric pH sensing maybe: (1) dyes that exhibit dual excitation wavelengths (corresponding toacid and conjugate base forms) and single emission wavelengths (e.g.,HPTS dyes); (2) single excitation wavelengths and dual emissionwavelengths (acid and base forms); or (3) dual excitation - dualemission dyes. Some dyes, such as the SNARF or SNAFL dyes may have bothdual-emission and dual-excitation properties. However a dual-dual dye,e.g., SNARF can be used as a single-dual or a dual-single.

Dual emission fiber-optic sensors based on seminapthofluorescein andcarboxynaphthofluorescein have been described that rapidly and reliablycorrelate intensity ratios to pH. See e.g., respectively, Xu, Z., A.Rollins, et al. (1998) “A novel fiber-optic pH sensor incorporatingcarboxy SNAFL-2 and fluorescent wavelength-ratiometric detection”Journal of Biomedical Materials Research 39: 9-15, and Song, A., S.Parus, et al. (1997) “High-performance fiber-optic pH microsensors forpractical physiological measurements using a dual-emission sensitivedye” Analytical Chemistry 69: 863-867. The extensive photobleachingobserved for these dyes may be accounted for by the ratiometricapproach, but it would still limit the useful lifetime of the sensor.

The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulphonic acid trisodiumsalt (HPTS) consists of a pyrene core with three sulfonic acid groupsand a hydroxyl group that imparts pH sensitivity around a pKa ofapproximately 7.3 (Wolfbeis, O. S., E. Fuerlinger, et al. (1983).“Fluorimetric analysis. I. Study on fluorescent indicators for measuringnear neutral (‘physiological’) pH values.” Fresneius' Z. Anal. Chem.314(2): 119-124); Wolfbeis et al. also have several patents onimmobilized HPTS. Yafuso and Hui describe another immobilizedfluorescent dye pH sensor in U.S. Pat. No. 4,886,338; incorporatedherein in its entirety by reference thereto. HPTS exhibits twoexcitation wavelengths, one at 405 nm and one at 457 nm, that correspondto the acid and its conjugate base (Agayn, V. I. and Dr. R. Walt (1993).“Fiber-optic sensor for continuous monitoring of fermentation pH.”Biotechnology 72(6):6-9). The subsequent pH-dependent shift inexcitation maximum about the pKa of 7.3 enables dual-excitation/singleemission ratiometric detection in the physiological range. This,together with a low toxicity (Lutty, G. A. (1978). “The acuteintravenous toxicity of stains, dyes, and other fluorescent substances.”Toxical Pharmacol. 44: 225-229) and insensitivity to oxygenconcentration (Zhujun, Z. and W. R. Seitz (1984). “A fluorescence sensorfor quantifying pH in the range from 6.5 to 8.5.” Analytical ChimicaActa 160: 47-55), makes HPTS a suitable probe for physiological andbioprocess pH measurements.

The presence of the three strongly anionic sulphonic acid groups allowsfor HPTS to be immobilized by ionic binding to cationic supports. Todate, covalent attachment of HPTS has been via sulfonamide coupling(U.S. Pat. No. 4,798,738). While effective in immobilizing the dye andpreserving pH sensitivity, polymer substrates are limited to those thatcontain primary amines. In addition, amine groups which remain on thesubstrate after coupling will affect the local pH inside the polymermatrix. The dye has been covalently attached to controlled pore glass(Offenbacher, H., O. S. Wolfbeis, et al. (1986). “Fluorescence opticalsensors for continuous determination of near-neutral pH values.” SensorActuator 9: 73-84) and aminoethyl cellulose (Schulman, S. G., S. Chen,et al. (1995). “Dependence of the fluorescence of immobilized1-hydroxypyrene-3,6,8-trisulfonate on solution pH: extension of therange of applicability of a pH fluorosensor.” Anal Chim Acta 304:165-170) in the development of fluorescence-based pH sensors thatoperate in neutral and acidic environments, as well as an intravascularblood gas monitoring system where it was used for both pH and pCO₂detection (Gehrich, J. L., D. W. Lubbers, et al. (1986). “Opticalfluorescence and its application to an intravascular blood gasmonitoring system.” IEE TBio-med Eng BME-33: 117-132). Fiber-optic pHsensors have been described with HPTS bound to an anion exchangemembrane (Zhujun, Z. and W. R. Seitz (1984)) or resin (Zhang, S., S.Tanaka, et al. (1995). “Fibre-optical sensor based on fluorescentindicator for monitoring physiological pH values.” Med Biol Eng Comput33: 152-156) and fixed to the tip of the optical fiber.

For example U.S. Pat. No. 5,114,676 (incorporated by reference herein inits entirety) provides a pH sensor with a fluorescent indicator whichmay be covalently attached to a particle or to a microcrystallinecellulose fiber. The sensor comprises an optically transparentsubstrate, a thermoplastic layer and a hydrogel. Part of the particlewith the indicator attached thereto is imbedded in a thermoplastic layerthat is coated on the substrate and mechanically adhered using heat andpressure. The majority of the particle/indicator is imbedded within ahydrogel layer that is applied over the thermoplastic layer. The pHsensor is applied to the tip of an optical waveguide.

Furthermore, with the recent availability of low cost UV LEDs, the dyecan be measured with relatively inexpensive instrumentation thatcombines UV and blue LEDs and a photodiode module. Such a setup has beendescribed (Kostov, Y., P. Harms, et al. (2001). “Low-costmicrobioreactor for high-throughput bioprocessing.” Biotechnol Bioeng72: 346-352) to detect the pH of a high throughput microbioreactorsystem via HPTS directly dissolved in the fermentation media.

In one embodiment of the present invention, the preferred sensing devicecomprises at least one light source, a detector, and a sensor comprisinga fluorescent reporter dye system. In one embodiment, the fluorescentreporter dye system comprises a fluorescent dye operably coupled to ananalyte-binding quencher. The dye may be covalently bound to thequencher or merely associated with the quencher. The dye and quencherare preferably operably coupled, which means that in operation, thequencher is in close enough proximity to the dye to interact with andmodulate its fluorescence. In one embodiment, the dye and quencher maybe constrained together within an analyte-permeable hydrogel or otherpolymeric matrix. When excited by light of appropriate wavelength, thefluorescent dye emits light (e.g., fluoresces). The intensity of thelight is dependent on the extent of quenching which varies with theamount of analyte binding. In other embodiments, the fluorescent dye andthe quencher may be covalently attached to hydrogel or other polymericmatrix, instead of to one another.

In one embodiment, a separate pH indicator dye is combined with adifferent dye that is functionalized with an analyte-binding moiety,such that the two dye system are immobilized together (e.g., in ahydrogel) in the sensor.

Some fluorescent pH indicator molecules absorb light at a particularwavelength and emit light at a second, longer wavelength. Their pHindicating function typically involves protonation and deprotonation.This means that these fluorescent pH indicators include a hydrogen atom(proton, H⁺) which forms part of the molecule (is bound to the molecule)in one pH range, but within another pH range the proton is dissociatedfrom the molecule. When the proton is disassociated from the molecule,the molecule takes on a negative charge, which is balanced by apositively-charged ion (e.g., Na⁺) in solution with the indicator. Thisarrangement is illustrated by Equation 1. R—H⇄R⁻H⁺

Where R represents a fluorescent molecule, it generally will exhibitfluorescence at a different wavelength (will be visible as a verydifferent color) based upon whether it is in the R—H form or in the R⁻form. For most molecules represented by R, this change will occurgenerally quite abruptly within a very narrow pH range, allowing R toserve as a very simple and reliable pH indicator. When placed insolution, it will exhibit one very distinct color (a color associatedwith its R—H form), and another very distinct color associated with itsR⁻.

For example, 8-Hydroxyl-1,3,6-pyrenetrisulphonate (HPTS) has beenconsidered one of the best potential indicators for pH determinationbecause of its excellent photo-stability, high quantum yield, dualexcitation, large Stokes' shift and long fluorescence emission. Adesirable feature of this indicator is that the acidic (associated HPTSform) and basic (dissociated PTS⁻) forms have different excitationwavelengths at 406 and 460 nm, with an isosbestic point at 418 nm, butexhibit a similar fluorescence emission maximum at 515 nm. The dualexcitation and single emission make HPTS suitable for ratiometricdetection of pH. The fluorescence intensity at 406 nm for the acid formdecreases but the intensity at 460 nm for the base form increases as thepH is raised accompanying the conversion of the acidic into basic formsof the dye.

Due to the hydroxyl (—OH) group on dyes such as HPTS and itsderivatives, these dyes are sensitive to the pH changes in theenvironment. The pH-dependent ionization of the hydroxyl group causesthese pyranine derivatives to have a pH-dependent absorption spectrawith different absorption maxima in its acidic form and basic form. Thefirst absorption maximum is the first excitation wavelength and thesecond absorption maximum is the second excitation wavelength. Theamounts of light absorbed by the fluorescent dye at the first excitationwavelength and the second excitation wavelength depend on or relate tothe pH of the medium the fluorescent dye is in contact with. The amountof light emitted by the dye (e.g., the fluorescent emission) at theemission wavelength depends on the amount of light absorption when thedye is irradiated at the excitation wavelength. Since the absorption isaffected by the pH of the medium, the fluorescent emission is alsoaffected by the pH. This provides the basis for the pH determinationwhile being able to measure the polyhydroxyl compound concentration.

In one preferred embodiment of the present invention, ratiometric pHsensing is accomplished using an optical sensor comprising at least oneexcitation light source operably coupled to the proximal end region ofan optical fiber, wherein the fiber has disposed along its distal endregion within the light path of the fiber, an indicator systemconfigured to generate a detectable emission signal in response to theexcitation light. Preferred embodiments of the sensor further compriseoptical means for sending the emission signal to a detector. Suchoptical means are well known in the art, and may involve e.g., a mirrorto return light, filters, lens, beam splitters, and optical fiberbundles and split configurations.

In preferred embodiments, the indicator system comprises a fluorophorethat exhibits at least two different forms and a pH-dependent shiftbetween these different forms, wherein this shift can be detected as achange in the emission intensity at a single wavelength or at twodifferent wavelengths. For example, one indicator system for ratiometricpH sensing comprises an fluorescent dye (e.g., HPTS) that absorbs lightat two different wavelength maxima's (λ_(acid) and λ_(base)) dependingon whether the dye is in its acid or base forms, and it emits light at asingle longer emission wavelength. More particularly, as pH isincreased, HPTS shows an increase in absorbance corresponding to theλ_(base) and a decrease in absorbance corresponding to the λ_(acid).These changes are due to the pH-dependent ionization of the hydroxylgroup. The emission spectrum for HPTS is independent of pH, with a peakemission wavelength of about 511 nm, but the intensity of the emittedlight depends on the amount of light absorbed (which varies with pH andthe excitation wavelength). So for example, if one excites HPTS at agiven pH with light of a first wavelength (e.g., λ_(acid)), one canmeasure the emission intensity at the single emission wavelength; theintensity will depend on the form of the dye (i.e., degree ofionization—which depends on the pH). One can also excite at a secondwavelength (e.g., λ_(base)) and measure the emission intensity at thesame given pH. The ratio of the emission intensities relates to the pHand is independent on the amount of the dye as well as certain opticalartifacts in the system. It is noted that any excitation wavelengths maybe used for the ratiometric sensing, but the λ_(acid) and λ_(base) arepreferred in accordance with one embodiment of the invention. Thewavelength at which the absorption is the same for the acid and baseforms of the dye is called the isobestic point-excitation at thiswavelength (I_(iso)) may also be used in ratiometric sensing inaccordance with other preferred variations to the invention. When aratio of emission intensities (e.g., I_(base)/I_(iso) orI_(base)/I_(acid)) is plotted against pH, a standard or calibrationcurve is generated (See e.g., FIGS. 3, 5 and 9). The ratiometric methodis similar regardless of whether the dye used is a dual exciter-singleemitter (like HPTS), or a single exciter—dual emitter, or a dualexciter—dual emitter, as long as the dye undergoes a pH sensitive shiftin form that yields a detectable change in spectral property.

Optical Glucose Sensing

Indicator systems comprising fluorescent dyes, including HPTS and itsderivatives, have been used in analyte detection. See e.g., U.S. Pat.Nos. 6,653,141, 6,627,177, 5,512,246, 5,137,833, 6,800,451, 6,794,195,6,804,544, 6,002,954, 6,319,540, 6,766,183, 5,503,770, and 5,763,238;and co-pending U.S. patent application Ser. Nos. 11/296,898 and60/833,081; each of which is incorporated herein in its entirety byreference thereto. In particular, details related to some preferredfluorescent dyes, quenchers/analyte binding moieties, and methods foroptically determining polyhydroxyl compound concentrations are disclosedin U.S. Pat. Nos. 6,653,141 and 6,627,177, and U.S. patent applicationSer. Nos. 11/296,898 and 60/833,081.

Device for Intravascular Determination of pH and Glucose

In one embodiment, the method and sensor monitor the pH of the media andthe concentration of analyte in vitro. In another embodiment, the methodand sensor monitor pH and analyte concentration in vivo. In anotherembodiment, the measured pH value can also be used to more correctlydetermine glucose concentration in vitro or in vivo. Specifically, thesimultaneous measurement of the pH value and the glucose concentrationwould enable real time correction of the signal of glucose response.Although it will be appreciated that the device in accordance with someembodiments comprise a sensor that may be used only to determine pH oranalyte (correction of which for pH may be done by conventional twosensor technologies or by testing the blood pH in vitro).

One embodiment provides a device for determining pH and theconcentration of a polyhydroxyl compound simultaneously, comprising asensor comprising a fluorescent dye operably coupled to a quencher;means for delivering one or more excitation wavelengths to said sensor;and means for detecting fluorescence emission from said sensor.

Another embodiment provides a device for determining the pH and thepolyhydroxyl compound concentration in a physiological fluid, comprisinga water-insoluble polymer matrix, wherein said polymer matrix ispermeable to polyhydroxyl compound; a fluorescent dye associated withsaid polymer matrix, wherein the fluorescent dye is configured to absorblight at a first excitation wavelength and a second excitationwavelength, and to emit light at an emission wavelength; a quenchercomprising an aromatic boronic acid substituted viologen, adapted toreversibly bind an amount of polyhydroxyl compound dependent on thepolyhydroxyl compound concentration, wherein said quencher is associatedwith said polymer matrix and operably coupled to the fluorescent dye,and wherein the quencher is configured to reduce the light intensityemitted by said fluorescent dye related to the amount of boundpolyhydroxyl compound; at least one excitation light source; and anemission light detector.

In one aspect, the present invention comprises a class of fluorescencequenching compounds that are responsive to the presence of polyhydroxylcompounds such as glucose in aqueous media at or near physiological pH.In other words, the quenching efficiency is controlled by theconcentration of these compounds in the medium. Preferred quencherscomprise a viologen substituted with at least one boronic acid groupwherein the adduct is immobilized in or covalently bonded to a polymer.The quencher, dye and polymer may also be covalently bonded to eachother. In another aspect, the present invention comprises a class offluorescent dyes which are susceptible to quenching by theviologen/boronic acid adduct.

The fluorescent dye and quencher are operably coupled to each other forpolyhydoxyl compound sensing. The dye and quencher may be linked througha polymer backbone in some embodiments. In other embodiments, the dyeand quencher could be in close proximity to each other for the quenchingof the fluorescent dye to occur, thereby reducing the fluorescentemission of the dye. When polyhydroxyl compound (e.g., glucose) binds tothe boronic acid to form boronate ester, the boronate ester interactswith the viologen and alters its quenching efficacy according to theextent of polyhydroxyl compound binding. As a result, the intensity offluorescent emission increases as more polyhydroxyl compounds are bondedto the quenchers.

In one preferred embodiment, the device comprises an optical fibercomprising a cavity disposed therein and having immobilized within thecavity an indicator system as described above (e.g., a fluorophoreoperably coupled to a glucose binding moiety/quencher and animmobilizing polymeric matrix). The device further comprises a lightsource and a detector.

Methods for Simultaneous Determination of pH and Glucose

One embodiment provides a method for determining the pH and thepolyhydroxyl compound concentration with one fluorescent dye, comprisingproviding a sensor comprising a fluorescent dye operably coupled to aquencher; contacting said sensor with a sample; irradiating said sensorat the first excitation wavelength; detecting a first fluorescenceemission of said sensor at an emission wavelength; irradiating saidsensor at the second excitation wavelength; measuring a secondfluorescence emission of said sensor at said emission wavelength;comparing the ratio of the first and second emissions with a pHcalibration curve to determine the pH of the sample; correlating theemission quenching with a standard curve at the known pH to determinethe polyhydroxyl compound concentration in said sample. Of course otheralgorithms are known for ratiometric pH sensing and may be used inaccordance with embodiments of the present invention. A controller, suchas a computer or dedicated device, may be used in some embodiments tocontrol the operations, including application of the excitation light,monitoring of detector signals, determining ratios, correlating ratioswith calibration curves, correlating glucose signals with standardcurves, correcting for pH changes, running routine sensor calibrationoperations, prompting operator actions, integrating user data input(e.g., finger stick glucose measurements) as programmed to maintainaccuracy, etc.

With respect to FIG. 1, a sensing device 100 in accordance with oneembodiment of the present invention comprises at least one light source11 (e.g., an excitation light source), a detector 15 (e.g., an emissionlight detector), and a sensor 13 comprising a fluorescent dye operablycoupled to a quencher and an optional polymer matrix. In someembodiments, the light source 11 may be adapted to selectively delivertwo or more different wavelength for the excitations of fluorescentdyes. This type of light source may be a tunable light source. In otherembodiments, one or more light sources may be used in conjunction withan optical filter 12 for attenuating the wavelengths. In otherembodiments, more than one light source 11 may be used to deliverdifferent excitation wavelengths. Such light source is also a means fordelivering a first and a second excitation wavelengths to the sensor.

The sensor 13 comprises a fluorescent dye that is sensitive to both thepH and the polyhydroxyl compound (e.g., sugar or glucose) concentrationof the medium when the dye is operably coupled to a quencher. Suchfluorescent dye exhibits a shift in excitation wavelength maximum with acorresponding shift in pH of the local environment of the fluorescentdye. As the pH of the local environment changes, the absorption at afirst excitation wavelength may increase, while the absorption at asecond excitation wavelength decreases, or vice versa. The change inabsorption at a selected wavelength can affect the level of fluorescenceemission, therefore ultimately permitting pH detection. The pH detectionis independent of the concentration of the polyhydroxyl compound in theenvironment. A suitable fluorescent dye is also susceptible to quenchingby molecules such as viologens. When the fluorescent dye is operablycoupled to a quencher (e.g., a viologen), the fluorescence emission isattenuated. The quencher may have an aromatic boronic acid moiety thatis capable of providing glucose recognition. The boronic acid reactsreversibly with glucose in aqueous media to form boronate ester, and theextent of such reaction is related to the glucose concentration in themedium. As more glucose is available to react with the quencher, thequencher's ability to accept electron from the dye decreases. As aresult, the attenuation of fluorescence emission by the quencher isdependent on the concentration of the polyhydroxyl compound (e.g.,glucose) to be detected.

A detector 15 is used to detect the fluorescent emission and inpreferred embodiments, may be linked to the electronic control 20 foranalysis. Optical filters, e.g., 14, can be placed between the sensor 13and the detector 15 for wavelength selection. Other optical componentsmay also be utilized, e.g., mirrors, collimating and/or focusing lenses,beam splitters, etc. Optical fibers can be used to deliver selectedwavelengths to the sensor and to deliver the fluorescence emission fromthe sensor to the detector. The light source and the detector may becontrolled by electronic control 20 such as a computer.

One embodiment of this invention provides a method for measuring pH andpolyhydroxyl compound concentration with a single fluorescent dye.Measurements can be carried out in vitro or in vivo. It may be necessaryto calibrate the sensor prior to performing the first measurement. Thismay be done by first acquiring the absorbance spectra of the sensor atvarious pHs to determine the wavelengths where isobestic point andabsorption maxima for acid and base forms occur and then acquiring theemission signals from at least two of these wavelengths at at least oneknown pH and glucose concentration.

For the pH and polyhydroxyl concentration measurements, the sensor 13 isfirst placed in contact with a sample. The sensor 13 is then irradiatedat the first excitation wavelength followed by the second excitationwavelength. The first and second excitation wavelengths are typicallychosen near the wavelength of the absorption maximum for the acidic formof the fluorescent dye (λ_(acid)), the wavelength of the absorptionmaximum for the basic form of the fluorescent dye (λ_(base)), or thewavelength of the isobestic point (λ_(iso)), or other selectedwavelength. The ratio of the emissions from the first and secondexcitation wavelengths are used to determine the sample pH. Either thefirst or second emission, once corrected for pH, can be used todetermine the sample glucose concentration.

In variations to the sensing device shown in FIG. 1, the detector may bea standard photodiode detector. There may be two diode detectors, onefor a reference and one for the emission signal. Instead of diodedetectors, the optical fiber carrying sensor output (fluorescentemission and/or reflected excitation light) may provide input directlyto a spectrophotometer or microspectrometer. In a preferred embodiment,the detector comprises a microspectrometer such as the UV/VISMicrospectrometer Module manufactured by Boehringer Ingelheim.

FIG. 2 shows one embodiment of an optical system that may be used inaccordance with preferred aspects of the present invention. Withreference to FIG. 2, certain embodiments comprise at least two lightsources, 301A and 301B. The light sources generate excitation light thatmay be transmitted (as illustrated) through collimator lenses 302A and302B. In certain embodiments, the resulting light from collimator lensesmay be transmitted (as illustrated) to interference filters 303A and303B. In certain embodiments, the resulting light from interferencefilters may be focused (as illustrated) by focusing lenses 304A and 304Binto fiber optic lines 305A and 305B. In certain embodiments, fiberoptic lines merge into a single fiber 306 that is continuous with thesensor 307, having the imbedded indicator system 307A. Thecross-sections of the fibers may vary (as illustrated) from a bundle offibers surrounding a central optical fiber 306A to a single fiber 307A.

In certain embodiments (as illustrated), the emission light signalsgenerated by the indicator system 307A as well as the excitation lightsignals are reflected by mirror 308 and transmitted back out of thesensor into the fiber optic outlet lines 309 and 309A. In theillustrated system, the outlet lines are augmented by including twointerference filters 312A, 312B and two detectors 313A, 313B. Inpreferred embodiments, the interference filter 312A is configured toblock the excitation light and allow the emission light to pass todetector 313A where the emission light is detected. In certainembodiments, the signal produced by the detector 313A is amplified bythe amplifier 314A and converted into a digital signal byanalog-to-digital converter 315A and transmitted to computer 316. Incertain embodiments, the interference filter 312B is configured to blockthe emission light and allow the excitation lights to pass to detector313B where the excitation light is measured. In certain embodiments, thesignal produced by the detector 313B is amplified by the amplifier 314Band converted into a digital signal by analog-to-digital converter 315Band transmitted to computer 316. Ratiometric calculations may beemployed to substantially eliminate or reduce non-glucose relatedfactors affecting the intensity of the emission light; these methods aredisclosed in detail in co-pending U.S. Provisional Application No.60/888,477, entitled “Optical systems and methods for ratiometricmeasurement of glucose using intravascular fluorophore sensors,” filedherewith on the same day, and incorporated herein in its entirety byreference thereto.

EXAMPLES Example 1

FIG. 3 shows an example of the excitation/absorption spectrum of afluorescent dye, in this case HPTS. From the absorption spectra of thefluorescent dye acquired at different pHs, λ_(acid), λ_(base) andλ_(iso) can be determined. At a lower pH (e.g., more acidic condition),the peak at around 405 nm is higher than the peak at around 460 nm, andis therefore the absorption maximum for the acidic form of thefluorescent dye. At a higher pH (e.g., more basic condition), the peakat round 460 nm is higher than the peak at around 405 nm, therefore isthe absorption maximum for the basic form of the fluorescent dye. Theλ_(iso) would be the wavelength where the absorption is independent ofthe pH, and it would be, for example, around 422 nm for HPTS.

The first fluorescence emission intensity (I_(x), which could beI_(acid), I_(base) or I_(iso)) at a emission wavelength, resulting fromthe irradiation at the first excitation wavelength (e.g., λ_(acid),λ_(base) or λ_(iso)), is then measured by the detector and the result isstored in the electronic control. Then the sensor is again irradiated atthe second excitation wavelength. The second excitation wavelength isdifferent from the first excitation wavelength and can also be selectedfrom λ_(acid), λ_(base) or λ_(iso). The detector will thendetect/measure the second fluorescence emission intensity (I_(y), whichcould be I_(acid), I_(base) or I_(iso)) resulting from the irradiationat the second excitation wavelength (e.g., λ_(acid), λ_(base) orλ_(iso)). The ratio of the first and the second fluorescence emissions(I_(x)/I_(y)) can then be computed. Since the I_(x)/I_(y) is independentfrom the polyhydroxyl concentration, a pH standard curve (I_(x)/I_(y)vs. pH) can be plotted without considering the effect of polyhydroxylconcentration.

Example 2 (HPTS/MABP4)

FIG. 4 shows independence of ratiometric pH sensing using HPTS/MABP4using the I_((base))/I_((iso)) ratio from glucose concentration. Thestructure of MABP4 is:

The data are plotted as a ratio of the fluorescence emission forcorresponding to excitation at 454 nm (base) and 422 nm (isobesticpoint) vs. pH in various glucose concentrations. The changes in glucoseconcentrations have no discernable effects on the value ofI_(base)/I_(iso) at each specific pH. Thus the pH of the sample can bemeasured using a standard curve of I_(x)/I_(y) vs. pH, regardless of thepolyhydroxyl compound concentration in the sample. By correlating orcomparing the measured I_(x)/I_(y) to the standard curve, one maydetermine the pH of the sample being measured.

FIG. 5 shows glucose response curves for HPTS/MABP4 excited at 422 nm(isobestic point) at different pHs. By plotting the ratio of I_(x)/I_(y)at various glucose levels (I) to I_(x)/I_(y) at zero glucoseconcentration (I₀) vs. glucose concentration, a standard polyhydroxylresponse curve can be used to determine the glucose concentration in asample from measured I/I₀ values. However, since I/I₀ value is dependenton the pH of the sample, the standard glucose response curve can beaffected by the different pH. To circumvent this, several standardglucose response curves at different pHs within the physiological rangecan be plotted and available for selection by either the electroniccontrol or the operator of the sensor device. When the I_(x)/I_(y)measurement of the sample is available, the electronic control or theoperator would know the pH of the sample from the standard I_(x)/I_(y)vs. pH curve, and the correct standard polyhydroxyl response curve(e.g., glucose response curve) may be used for determining the accurateglucose concentration. Although the examples shown in the figuresconcern determination of glucose concentration, the application of themethod and device of the present invention is not limited to detectingglucose concentration. Since the fluorescent system responds topolyhydroxyl compounds the same way it responds to glucose, the sensordevice can be used to detect any polyhydroxyl compound concentration andthe pH at the same time.

Example 3 (SNARF-1)

FIG. 6 shows the absorption spectra of SNARF-1 at different pHs insolution. SNARF is a tradename for a class of commercial dyes fromMolecular Probes, Inc. These experiments were carried out using SNARF-1.FIGS. 7 and 8 show glucose response curves for SNARF-1/3,3′-oBBV insolution at different pHs determined at 514 nm excitation/587 nmemission (FIG. 7), or at 514 nm excitation/625 nm emission (FIG. 8).FIG. 9 shows ratiometric sensing of pH at different glucoseconcentrations with SNARF-1/3,3′-oBBV in solution using theI_((base))/I_((acid)) ratio determined at a single excitation wavelengthof 514 nm and emission wavelengths of 587 and 625 nm. Thus, thedual-dual dye SNARF-1 may be used operably coupled to the quencher3,3′-oBBV (in solution) as a single exciter-dual emitter fluorophore todetermine both pH ratiometrically and glucose.

Example 4 (HPTS-triLysMA/3,3′-oBBV/DMAA)

FIG. 10 shows the glucose response of HPTS-triLysMA/3,3′-oBBV/DMAAindicator system at different pHs. FIG. 11 shows ratiometric sensing ofpH at different glucose concentrations with theHPTS-triLysMA/3,3′-oBBV/DMAA indicator system, using theI_((base))/I_((acid)) ratio. It can be seen that this indicator systemprovides a linear pH curve over the physiologic pH range.

Example 5 (HPTS-triCysMA/3,3′-oBBV/DMMA)

FIG. 12 shows ratiometric sensing of pH at different glucoseconcentrations with the HPTS-triCysMA/3,3′-oBBV/DMMA indicator system,using the I_((base))/I_((acid)) ratio. It can be seen that thisindicator system provides a linear pH curve over the physiologic pHrange. For this example, the indicator system was immobilized in ahydrogel embedded at the end of an optical fiber. The acid and baseemission signals were measured using a hand-held detector.

Lifetime Chemistry

In another preferred embodiment, glucose concentrations can bedetermined by exploiting the phenomena of fluorescence resonance energytransfer (FRET). FRET is the transfer of energy from a donor fluorophoreto an acceptor molecule. FRET occurs when the donor fluorophore, whichfluoresces at a wavelength absorbed at least in part by the acceptormolecule, is in close proximity to the acceptor such that the donorfluorophore can transfer energy to the acceptor through molecularinteractions. The fluorescence lifetime of the fluorophore, where thefluorescence lifetime is the time the fluorophore remains in the excitedstate, is altered by FRET. Thus, measuring the fluorescence lifetime ofthe fluorophore allows one to determine whether the fluorophore is boundto the acceptor.

Lifetime can be measured by using a time-domain method where thefluorophore is excited by a brief pulse of excitation light and thefluorescence intensity is measured over time. The excitation pulse canbe a pulse from a laser with a duration in the picoseconds range up to aduration of about a few nanoseconds. In other embodiments, the pulseduration can be greater than about a few nanoseconds. The fluorescenceintensity of the fluorophore as a function of time is given by theequation:

I(t)=I ₀*exp(−t/τ)   Equation 1

I(t) is the fluorescence intensity at time (t), I₀ is the initialintensity after excitation and τ is the fluorescence lifetime which isdefined as the time required for I(t) to decay to I₀/e. Equation 1 isapplicable to a fluorophore with a single exponential decay offluorescence and a lifetime that is substantially longer than theexcitation pulse. FIG. 13 shows a graph of the decay of the fluorescentemission 400 over time after a pulse of excitation light 402. The timeit takes the initial intensity, I₀, to drop to I₀/e is equal to thelifetime, τ.

An alternative method of measuring lifetime is by a frequency-domainmethod where the fluorophore is excited by a frequency modulatedexcitation light. The fluorescence lifetime, τ, can be determined bymeasuring the phase shift of the emission from the fluorophore relativeto the excitation light, or by measuring the modulation ratio, using thefollowing equations:

τ_(φ)=ω⁻¹*tan(φ)   Equation 2

ω=2πƒ  Equation 3

τ_(M)=ω⁻¹*(M ⁻²−1)^(1/2)   Equation 4

$\begin{matrix}{M = \frac{\left( {{AC}/{DC}} \right)_{EM}}{\left( {{AC}/{DC}} \right)_{EX}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

τ_(φ) is the lifetime determined by measuring the phase shift, φ. ω isthe angular frequency of the frequency modulated excitation light and ƒis the linear frequency. τ_(M) is the lifetime determined by measuringthe modulation ratio, M. AC is the magnitude of the alternating portionof the signal, or the amplitude of the wave, while DC is the amplitudeof the DC portion of the signal. EM refers to the emission signal, andEX refers to the excitation signal. FIG. 14 is a graph showing therelationship between the emission signal 500 and the excitation signal502 and the variables described in Equations 2-5.

Preferred binding assay configurations for use in the sensor include areversible competitive, reagent limited, binding assay, the componentsof which include an analyte analog and an analyte binding agent capableof reversibly binding both the analyte of interest and the analyteanalog. The analyte of interest and the analyte analog compete forbinding to the same binding site on the analyte binding agent. Suchcompetitive binding assay configurations are well known in the art ofclinical diagnostics and are described, by way of example, in TheImmunoassay Handbook, ed. David Wild, Macmillan Press 1994. Suitableanalyte binding agents for use in the assay would include antibodies orantibody fragments which retain an analyte binding site (e.g. Fabfragments), lectins (e.g. concanavalin A), hormone receptors, drugreceptors, aptamers and molecularly-imprinted polymers. Preferably theanalyte analog should be a substance of higher molecular weight than theanalyte such that it cannot freely diffuse out of the sensor. Forexample, an assay for glucose might employ a high molecular weightglucose polymer such as dextran as the analyte analog.

Suitable optical signals which can be used as an assay readout inaccordance with the invention include any optical signal which can begenerated by a proximity assay, such as those generated by fluorescenceresonance energy transfer, fluorescence polarisation, fluorescencequenching, phosphorescence technique, luminescence enhancement,luminescence quenching, diffraction or plasmon resonance.

In some preferred embodiments of the sensor of the inventionincorporates a competitive, reagent limited binding assay whichgenerates an optical readout using the technique of fluorescenceresonance energy transfer. In this assay format the analyte analog islabelled with a first chromophore and the analyte binding agent islabelled with a second chromophore. One of the first and secondchromophores acts as a donor chromophore and the other acts as anacceptor chromophore. It is an important feature of the assay that thefluorescence emission spectrum of the donor chromophore overlaps withthe absorption spectrum of the acceptor chromophore, such that when thedonor and acceptor chromophores are brought into close proximity by thebinding agent a proportion of the energy which normally would producefluorescence emitted by the donor chromophore (following irradiationwith incident radiation of a wavelength absorbed by the donorchromophore) will be non radiatively transferred to the adjacentacceptor chromophore, a process known in the art as FRET, with theresult that a proportion of the fluorescent signal emitted by the donorchromophore is quenched and, in some instances, that the acceptorchromophore emits fluorescence. Fluorescence resonance energy transferwill generally only occur when the donor and acceptor chromophores arebrought into close proximity by the binding of analyte analog to analytebinding agent. Thus, in the presence of analyte, which competes with theanalyte analog for binding to the analyte binding agent, the amount ofquenching is reduced (resulting in a measurable increase in theintensity of the fluorescent signal emitted by the donor chromophore ora fall in the intensity of the signal emitted by the acceptorchromophore) as labelled analyte analog is displaced from binding to theanalyte binding agent. The intensity or lifetime of the fluorescentsignal emitted from the donor chromophore thus correlates with theconcentration of analyte in the fluid bathing the sensor.

An additional advantageous feature of the fluorescence resonance energytransfer assay format arises from the fact that any fluorescent signalemitted by the acceptor chromophore following excitation with a beam ofincident radiation at a wavelength within the absorption spectrum of theacceptor chromophore is unaffected by the fluorescence resonance energytransfer process. It is therefore possible to use the intensity of thefluorescent signal emitted by the acceptor chromophore as an internalreference signal, for example in continuous calibration of the sensor orto monitor the extent to which the sensor has degraded and thus indicatethe need to replace the sensor. As the sensor degrades, the amount ofacceptor chromophore present in the sensor will decrease and hence theintensity of fluorescent signal detected upon excitation of the acceptorchromophore will also decrease. The fall of this signal below anacceptable baseline level would indicate the need to implant or inject afresh sensor. Competitive binding assays using the fluorescenceresonance energy transfer technique which are capable of being adaptedfor use in the sensor of the invention are known in the art. U.S. Pat.No. 3,996,345 describes immunoassays employing antibodies andfluorescence resonance energy transfer between a fluorescer-quencherchromophoric pair. Meadows and Schultz (Anal. Chim. Acta (1993 280:pp21-30) describe a homogeneous assay method for the measurement ofglucose based on fluorescence resonance energy transfer between alabelled glucose analog (FITC labelled dextran) and a labelled glucosebinding agent (rhodamine labelled concanavalin A). In all of theseconfigurations the acceptor and donor chromophores/quenchers can belinked to either the binding agent or the analyte analog.

Fluorescence lifetime or fluorescence intensity measurements may bemade. As described in Lakowitz et al, Analytica Chimica Acta, 271,(1993), 155-164, fluorescence lifetime may be measured by phasemodulation techniques.

In some preferred embodiments as shown in FIGS. 15A, 15B and 15C, acompetitive binding system to measure glucose using FRET comprises aglucose binding molecule 600 linked to a donor fluorophore 602 and aglucose analog 604 linked to an acceptor molecule 606. The glucosebinding molecule 600 is capable of binding with both glucose 608 and theglucose analog 604. As shown in FIG. 15A, when the glucose analog 604 isbound to the glucose binding molecule 600, the fluorescent emission 500from the fluorophore 602 is reduced in magnitude and shifted in phaseand lifetime by FRET 610 because the fluorophore 502 is in closeproximity to the acceptor 606. In other embodiments, the fluorophore 602is linked to the glucose analog 604 and the acceptor 606 is linked tothe glucose binding molecule 600.

As shown in FIG. 15B, glucose 608 competes with the glucose analog 604for the binding site on the glucose binding molecule 600. As shown inFIG. 15C, the glucose molecule 608 can displace the glucose analog 604from the glucose binding molecule 600 so that the acceptor 606 does notalter the emission lifetime 500 of the fluorophore 602 via FRET 610.

In a system where there are a certain concentration of glucose bindingmolecules, glucose analogs and glucose molecules, an equilibrium willexist between the number of bound glucose molecules to the number ofbound glucose analogs. A change in the number of glucose molecules inthe system, changes the equilibrium between bound glucose molecules tobound glucose analogs. This in turn changes the mean lifetime of thefluorophore emission.

In some preferred embodiments, the system is excited by a frequencymodulated excitation light less than approximately 1 MHz, betweenapproximately 1 to 200 MHZ, or greater than approximately 200 MHz. Insome embodiments, the frequency is approximately 0.05, 0.1, 1, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190 or 200 MHz. By measuring the degree of the phase shift of thesystem, an average FRET induced phase shift for the system can bedetermined which corresponds to an average lifetime value for the systemas defined by Equations 2 and 3 described above. Both the phase shiftand the lifetime values can be correlated to the glucose concentration.The magnitude of the phase shift is independent of the amplitude of theemission.

In other preferred embodiments, the system is excited by a pulse and thedecay of the fluorescence is measured over time. The lifetime can bedetermined using Equation 1 described above, and glucose concentrationcan be correlated to the lifetime value.

In preferred embodiments, the glucose binding molecule with a donorfluorophore and the glucose analog with an acceptor can be substantiallyimmobilized in the hydrogel described above such that diffusion of theglucose binding molecule and the glucose analog out of the hydrogel issubstantially reduced. In addition, the sensor is configured to provideexcitation light at a wavelength absorbed by the donor fluorophore asdescribed above. In some embodiments, the excitation light is providedas a short pulse from a laser or a light emitting diode (LED). In otherembodiments, the excitation light is frequency modulated. In someembodiments, the frequency modulated excitation light is provided by alaser. In some embodiments the frequency modulated excitation light isprovided by a LED. The sensor also has a detector that detects theamplitude of the emission over time and/or the phase shift of theemission and/or the amplitudes of the AC and DC portions of the emissionand excitation light. The detector can be a photodetector or multiplephotodetectors. The excitation and emission light can be transmittedthroughout the sensor via optical fibers.

In some embodiments, the sensor can be introduced into a patient's bloodvessel, such as a vein, artery or capillary, for measuring theintravascular concentration of an analyte in the patient's blood. Insome embodiments, the chemistry used to measure the concentration of theanalyte is based on a correlation of fluorescence intensity of afluorophore to analyte concentration, as described above in more detail.In some embodiments, the chemistry used to measure the concentration ofthe analyte is based on a correlation of fluorescence lifetime of afluorophore to analyte concentration, as described above in more detail.In some embodiments, the sensor is used to measure the concentration ofglucose.

While a number of preferred embodiments of the invention and variationsthereof have been described in detail, other modifications and methodsof using and medical applications for the same will be apparent to thoseof skill in the art. Accordingly, it should be understood that variousapplications, modifications, and substitutions may be made ofequivalents without departing from the spirit of the invention or thescope of the claims.

1. An intravascular sensor for determining an analyte concentration inblood comprising: an optical fiber comprising a sensor chemistry portioncapable of insertion into a blood vessel, the sensor chemistry portioncomprising: an analyte binding molecule capable of binding an analyte;and a fluorophore associated with the analyte binding molecule, thefluorophore having a first fluorescence when the analyte bindingmolecule is not bound to the analyte and a second fluorescence when theanalyte binding molecule is bound to the analyte; a light source; and adetector.
 2. The sensor of claim 1, wherein the first fluorescence is afirst fluorescence intensity and the second fluorescence is a secondfluorescence intensity.
 3. The sensor of claim 1, wherein the firstfluorescence is a first fluorescence lifetime and the secondfluorescence is a second fluorescence lifetime.
 4. The sensor of claim1, wherein the analyte is glucose.
 5. The sensor of claim 1, furthercomprising an optical fiber.
 6. The sensor of claim 1, wherein the lightsource is a laser.
 7. The sensor of claim 1, wherein the light source isa light emitting diode.
 8. The sensor of claim 1, wherein the sensorchemistry portion further comprises a hydrogel that substantiallyimmobilizes the analyte binding molecule and the fluorophore and ispermeable to the analyte.
 9. The sensor of claim 1, wherein the sensorchemistry portion further comprises a membrane wherein the analytebinding molecule and the fluorophore are substantially retained within avolume at least partially enclosed by the membrane.
 10. An intravascularsensor for determining an analyte concentration in blood comprising: asensor chemistry portion capable of insertion into a blood vessel, thesensor chemistry portion comprising: an analyte binding molecule capableof binding an analyte; and a fluorophore associated with the analytebinding molecule, the fluorophore having a first fluorescence intensitywhen the analyte binding molecule is not bound to the analyte and asecond fluorescence intensity when the analyte binding molecule is boundto the analyte; a light source; and a detector.
 11. An intravascularsensor for determining an analyte concentration in blood comprising: asensor chemistry portion capable of insertion into a blood vessel, thesensor chemistry portion comprising: an analyte binding molecule capableof binding an analyte; and a fluorophore associated with the analytebinding molecule, the fluorophore having a first fluorescence lifetimewhen the analyte binding molecule is not bound to the analyte and asecond fluorescence lifetime when the analyte binding molecule is boundto the analyte; a light source; and a detector.
 12. A method fordetermining the glucose concentration in blood comprising: providing thesensor of claim 1; inserting the sensor into a blood vessel; irradiatingthe fluorophore with an excitation signal; detecting a fluorescenceemission from the fluorophore; and determining the concentration ofglucose.
 13. The method of claim 12, wherein the excitation signal is apulse of light.
 14. The method of claim 13, further comprising measuringthe decay of the fluorescence emission over time; and calculating afluorescence lifetime.
 15. The method of claim 12, wherein theexcitation signal is frequency modulated.
 16. The method of claim 15,further comprising measuring the phase shift between the fluorescenceemission and the excitation signal.
 17. The method of claim 16, furthercomprising calculating a fluorescence lifetime.
 18. The method of claim15, further comprising measuring a modulation ratio; and calculating afluorescence lifetime.
 19. The method of claim 12, further comprisingmeasuring the fluorescence intensity of the fluorescence emission. 20.An intravascular sensor for determining an analyte concentration inblood, comprising an optical fiber comprising a sensor chemistry portiondisposed along a distal region of the optical fiber, wherein the sensorchemistry portion is sized and physiologically compatible with residingin a blood vessel, wherein the sensor chemistry portion is selected fromequilibrium fluorescence chemistry or lifetime chemistry.
 21. A methodfor determining the glucose concentration in blood comprising: providingthe sensor of claim 11; inserting the sensor into a blood vessel;irradiating the fluorophore with an excitation signal; detecting afluorescence emission from the fluorophore; and determining theconcentration of glucose.